Nucleolar protein B23/Nucleophosmin. Novel protein-protein interactions can be detected by co-immunoprecipitation of specific proteins with a target protein of interest. We analyzed proteins from Xenopus egg extracts that specifically bound to the Ulp1p-like SUMO proteases xSENP3 and 5. Analysis of gel separated proteins by tryptic digestion followed by LC/MS/MS allowed identification of several novel binding partners. A protein that strongly bound to xSENP3 was identified as the Xenopus B23/Nucleophosmin homolog. This binding was shown by further experiments (carried out in LGRD) to be essential for stable accumulation of SENP3 and SENP5 in mammalian tissue culture cells.[unreadable] [unreadable] Lipid Quantification in Serum. We have continued developing methodology to quantify cardiolipins in human serum by mass spectrometry. This effort is in association with a clinical study underway in the Institute to evaluate the effects of antibiotic treatment of pregnant women colonized with the Group B streptococcal (GBS) organism. The hypothesis of the study is that the typical peri-natal penicillin treatment gives rise to a large increase of circulating cardiolipins in the infant which then leads to respiratory distress. It has been demonstrated in other studies in newborn sheep that the GBS organisms secrete a specific cell wall membrane cardiolipin with penicillin treatment and that this substance causes respiratory distress at levels of about 100 pmole/mL in serum. It is not known whether the respiratory distress observed in a fraction of infants born to GBS colonized mothers is a result of a similar effect, or perhaps by a related effect caused by a release of endogenous cardiolipins stimulated by the bacterial death. The analytical approach involves the addition of an internal standard to a 200 uL serum sample, a combination of liquid-liquid and solid phase extractions, followed by an LC-MS analysis that incorporates an extraction/recovery standard to monitor system quality control. We have shown that cardiolipin can be extracted from serum with greater than 75% efficiency and that cardiolipin serum levels of less than 100 pmole/mL can be determined. We continue to refine the mass spectrometric methods for the determination of these materials in order to increase analytical robustness and sample throughput rate. [unreadable] [unreadable] Tubulin Post-translational Modifications. Continuing work on characterization of post-translational modifications of tubulins has recently yielded interesting results with respect to phosphorylation sites. A characteristic addition of 80Da to a chicken erythrocyte tubulin C-terminal CNBr digest fragment shows the presence of phosphorylation, but to date the modified residue has not been identified. In another study employing a kinease known to phosphorylate tubulin in a 2 mole phosphate to mole tubulin ratio, phosphorylation sites of HeLa tubulins have been identified. Two different beta-tubulins found in HeLa cells have been shown to phosphorylate, not in the C-terminal region, but in the main sequence of the proteins in a region known by X-ray crystallography to exist in a loop, external to the protofilaments, that plays an active role in microtubule assembly. To this point however, analogous sites on alpha-tubulins have not been identified. There may be substantial therapeutic implications to this observation, particularly in light of the presence or absence of phosphorylation in the similar region of the alpha tubulins, since this loop is known to be the site in which tamoxifen inhibits microtubule assembly.[unreadable] [unreadable] Improvement in Spectral Reliability. The efforts in development of methods to obtain more robust mass spectra of given samples by generating consensus spectra from multiple replicates have continued to be productive. When applied to peptide fragmentation spectra as part of an effort to infer the presence of proteins in gel samples, we have preliminary results showing a substantial improvement in results by using this approach. The effect of this approach is particularly significant when applied to peptide sequencing de novo. While our novel algorithm for deducing peptide sequences de novo often yields useful results for a particular peptide precursor being fragmented, generating a consensus spectrum from multiple replicate spectra of the same peptide precursor seems to give meaningful results every time. Encouraged by these results, we plan on extending the approach to routine protein inference from unknown samples in both MALDI TOF-TOF and LC/MS-MS methodologies.